Phosphoribosyl Anthranilate Isomerase Catalyzes a Reversible Amadori Reaction

Abstract

Data from steady state and transient kinetics show that the functional phosphoribosyl anthranilate isomerase domain of the naturally bifunctional enzyme from Escherichia coli has properties similar to those of its artificially excised domain. The naturally monofunctional enzyme from Saccharomyces cerevisiae has significantly higher values of both kcat and kcat/KMPRA. The primary product of a single turnover of phosphoribosylanthranilate is fluorescent, but it slowly isomerizes to the nonfluorescent stable product. The latter is the competent substrate of indoleglycerol phosphate synthase, which catalyzes the subsequent step of tryptophan biosynthesis. The isomerization is characterized by a monoexponential decay independent of phosphoribosyl anthranilate isomerase. Due to a tentative assignment of the fluorescent, primary product and the nonfluorescent, stable product to an enol and a keto compound, respectively, tryptophan biosynthesis appears to be rate-limited by an uncatalyzed enol/keto tautomerization. A formal kinetic mechanism of the reaction catalyzed by phosphoribosyl anthranilate isomerase is proposed that is consistent with the combined enzymic and ligand binding properties of the three variants of phosphoribosyl anthranilate isomerase.

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…This finding was corroborated by fluorescence titration studies with the product analogue reduced 1-(2-carboxyphenylamino)-1-deoxyribulose-5-phosphate (rCdRP), which yielded similar dissociation constants for HisAFcomIII and ecTrpF (Table 1) (25). Attempts to further increase the TrpF activity by performing another round of random mutagenesis and selection did not lead to the identification of variants with turnover numbers or Michaelis constants surpassing those of HisAFcomIII.…”

“…The TrpF activities of the purified HisAF and HisA variants were followed at 25°C by a fluorimetric assay and analyzed as described (14,25). The binding of the product analogue rCdRP to HisAFcomIII and HisA-II was followed by fluorescence energy transfer and analyzed as described (15,31).…”

A Study in Molecular Contingency: Glutamine Phosphoribosylpyrophosphate Amidotransferase is a Promiscuous and Evolvable Phosphoribosylanthranilate Isomerase

“…This value is 2.3 × 10 7 -fold below that of the wild-type E. coli PRAI (k cat /K M = 6.8 × 10 6 s −1 .M −1 ). 19 Our average yield of purified (His) 6 -PurF(1-04) was 19 mg.L −1 culture. Based on the cell density of the culture at the end of the induction (and the rather optimistic assumption that our purification was 100% efficient), it can be estimated that every over-expressing cell contained > 1.8 × 10 5 molecules of (His) 6 -PurF(1-04).…”

“…49 The catalytic efficiency of the wild-type PRAI falls squarely in this range (k cat /K M = 6.8 × 10 6 s −1 .M −1 ). 19 In contrast, we estimated the catalytic efficiency of our most efficient variant, (His) 6 -PurF(1-04), to be over seven orders of magnitude lower (k cat /K M = 0.3 s −1 .M −1 ). The E. coli strain over-expressing this variant is nevertheless similar in phenotype to wild-type cells (colony formation in ~30 h, c.f.…”

Directed evolution of a (βα) 8 -barrel enzyme to catalyze related reactions in two different metabolic pathways

“…Steady-state enzyme kinetics of the TrpF reaction was measured at 25°C by f luorescence spectroscopy according to ref. 18. Anthranilic acid was first converted into an equal amount of PRA by 0.7 M anthranilate phosphoribosyltransferase from yeast, using a large molar excess of phosphoribosyl pyrophosphate (PRPP).…”